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BMC Biotechnology
BioMed Central
Open Access
Methodology article
One-step selection of Vaccinia virus-binding DNA aptamers by
MonoLEX
Andreas Nitsche*1, Andreas Kurth1, Anna Dunkhorst1, Oliver Pänke2,3,
Hendrik Sielaff2, Wolfgang Junge2, Doreen Muth1, Frieder Scheller4,
Walter Stöcklein4, Claudia Dahmen5, Georg Pauli1 and Andreas Kage6
Address: 1Centre for Biological Safety 1, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany, 2Department of Biology/Chemistry, Division
of Biophysics, University of Osnabrueck, 49069 Osnabrueck, Germany, 3Biosystems Technology, University of Applied Sciences Wildau,
Bahnhofstr. 1, 15747 Wildau, Germany, 4Institute of Biochemistry and Biology, Department of Analytical Biochemistry, University of Potsdam,
Karl-Liebknecht-Str. 24-25, 14476 Golm, Germany, 5AptaRes AG, Im Biotechnologiepark TGZ I, 14943 Luckenwalde, Germany and 6Institute of
Laboratory Medicine and Pathobiochemistry, Charité Universitätsmedizin Berlin, Westend Haus 31, Spandauer Damm 130, 14050 Berlin,
Germany
Email: Andreas Nitsche* - [email protected]; Andreas Kurth - [email protected]; Anna Dunkhorst - [email protected];
Oliver Pänke - [email protected]; Hendrik Sielaff - [email protected]; Wolfgang Junge - [email protected];
Doreen Muth - [email protected]; Frieder Scheller - [email protected]; Walter Stöcklein - [email protected];
Claudia Dahmen - [email protected]; Georg Pauli - [email protected]; Andreas Kage - [email protected]
* Corresponding author
Published: 15 August 2007
BMC Biotechnology 2007, 7:48
doi:10.1186/1472-6750-7-48
Received: 6 February 2007
Accepted: 15 August 2007
This article is available from: http://www.biomedcentral.com/1472-6750/7/48
© 2007 Nitsche et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: As a new class of therapeutic and diagnostic reagents, more than fifteen years ago
RNA and DNA aptamers were identified as binding molecules to numerous small compounds,
proteins and rarely even to complete pathogen particles. Most aptamers were isolated from
complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection
and amplification steps. Here we report the application of a new one-step selection method
(MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism
for complex target structures.
Results: The selection against complete Vaccinia virus particles resulted in a 64-base DNA
aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon
Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer
blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia
virus and other orthopoxviruses in a concentration-dependent manner.
Conclusion: The MonoLEX method is a straightforward procedure as demonstrated here for the
identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single
affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and
a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the
selection of high affinity aptamers by reducing the competition between aptamers of different
affinities during the PCR step, indicating an advantage for the single-round MonoLEX method.
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Background
New intervention strategies are required to detect, prevent
and control disease outbreaks. In this context, orthopoxviruses (OPV) like Variola virus, Monkeypox virus and
bioengineered OPV are often discussed to be potentially
used as biological weapons [1]. Therefore there is general
consent that the establishment of a comprehensive pool
of antiviral drugs, including substances that inhibit OPV
replication by mechanisms other than the substances
already characterized, is a reliable strategy for treating and
preventing smallpox and other OPV infections in humans
[2].
Aptamers promise to be such additional candidates for
prophylaxis and treatment of infectious diseases, as they
can be directed against a wide variety of target molecules
like toxins or even complete microorganisms [3-7].
Aptamers are short DNA or RNA oligonucleotides and,
like antibodies, bind to their target by their three-dimensional structure with high affinity and specificity. Compared to antibodies, aptamers have several advantages, as
they are selected in vitro which also enables selection
against toxic or weakly immunogenic targets. DNA aptamers are heat and protease resistant without stabilizing
modifications. Due to chemical synthesis, aptamer production can easily be scaled up [3]. During the selection
process aptamers with the highest affinity to a target structure are isolated from a pool of oligonucleotides with random positions for nucleic acids, which, depending on the
length of the aptamers, contain up to 1015 different
sequence variants, representing a significantly larger pool
of variants than antibodies do. In addition to these evident benefits, aptamers exhibit some drawbacks, including their reduced resistance to nucleases and the problem
of delivery and clearance in therapeutic application. First
attempts to stabilize aptamers were promising [8,9].
The enrichment procedure that has commonly been used
after being first reported in 1990 was called Systematic
Evolution of Ligands by Exponential Enrichment (SELEX)
[10,11]. Since then, great efforts have been made to
improve this method [12,13], and many different aptamers have been selected by various SELEX-based protocols
for a wide variety of targets ranging from small molecules
to whole cells [14] and bacteria [15]. Some of these
aptamers were shown to possess inhibitory activity with
certain HIV strains [16], to block cell binding of human
cytomegalovirus (CMV) [17] or influenza virus hemagglutinin [18] or were selected as tools for differentiation of
closely related influenza strains [19]. Recently, the first
aptamer-based drug Macugen was approved by the FDA
for treating wet forms of macula degeneration, which is an
important step towards the acceptance of aptamers in
clinical therapy [20]. Further drug candidates are under
clinical trials [21]. Finally, an aptamer-based detection
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device for cocaine already in practical application has
been published recently [22].
As an alternative to the SELEX process with 7 to more than
30 selection and amplification cycles requiring large
amounts of the target molecule, a new selection process
was established. Based on reports about selecting functional oligonucleotides [23] and the potential of oligonucleotides as non-Watson-Crick-type binders to peptides
and proteins [24], the present study applied this new onestep aptamer isolation protocol (MonoLEX) to retrieve
DNA aptamers that have the potential to bind specifically
to OPV particles. The MonoLEX approach combined a single affinity chromatography step with subsequent physical segmentation of the affinity resin and one single final
exponential amplification step of bound aptamers. A
schematic comparison of SELEX and MonoLEX is given in
figure 1. Specific aptamers were selected from a combinatory library of oligonucleotides characterized by two
flanking primers of known sequence and an internal
region of 20 random nucleotide positions (N20 DNA
library). Under adequate chromatographic conditions,
like flow laminarity, sufficient capacity and homogeneity
of the resin, high-affinity-binding aptamers stuck to the
target, whereas weakly binding oligonucleotides could be
removed from the resin by ample washing. Since the selection is an in vitro process, depending on the final application of the aptamer, the selection conditions can be
adapted accordingly.
Results
Identification of high-affinity-binding aptamers by
MonoLEX
To select DNA aptamers specific for Vaccinia virus (VACV)
as a model for OPV, aptamer selection was performed in
two steps. An initial chromatography was performed on
proteins from virus-free cell culture supernatant to eliminate aptamers binding to cell debris or media components. Subsequently, a second chromatography was
carried out on resin-bound heat-inactivated VACV particles. For direct recovery of high-affinity-binding aptamers,
the resin of the affinity column was physically segmented
into slices and the amount of retained binding aptamers
was estimated by quantitative real-time PCR. High concentrations of aptamers not eluted during the extended
washing procedure were identified on several affinity column segments as indicated by the different grey and
colored bars in Fig. 2a. Indicated column fragments contained aptamer amounts that significantly differed from
the background. A subsequent fluorescence curve melting
analysis (FCMA) of the PCR products revealed clearly distinguishable single or double melting peaks for four
aptamers which are shown in color in Fig 2a. The different
melting temperatures observed for these four aptamers
indicate different nucleic acid composition of the oligo-
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Figure 1 presentation of MonoLEX in comparison to SELEX
Schematic
Schematic presentation of MonoLEX in comparison to SELEX. Based on a combinatory oligonucleotide library,
SELEX comprises several cycles of target binding, elution and amplification of putative aptamers. In contrast, MonoLEX starts
with one affinity chromatography to sort non-binding oligonucleotides, low-affinity aptamers and high-affinity aptamers. Highly
affine aptamers are amplified once and characterized further by an aptamer blot assay.
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nucleotides retained in distinct positions on the affinity
resin (Fig. 2b). As observed for the two aptamers A38 and
A77 (blue and green line in Fig 2a), more than one melting peak suggested the presence of at least two different
aptamers with different melting temperatures amplified
from one affinity column segment.
The sequences of these aptamers were determined by
pyrosequencing as described above. None of the aptamers
showed sequence homology to known viral or cellular
sequences in the variable region as proven by BLAST
search, neither including nor excluding the flanking
primer sequences.
To verify the binding specificity of the identified aptamers, several methods were applied. First, an aptamer blotting assay was performed binding aptamers to heatinactivated VACV. Bound aptamers were quantified using
real-time PCR, revealing a significant enrichment of
aptamer molecules as indicated by a CT-value shift of 10
(Fig. 2c, inset, red line) in relation to the background
without VACV (Fig. 2c, inset, black dotted line). Additional peaks seen in FCMA directly after amplification of
the aptamers 38 and 77 (A38 and A77, Fig. 2b) disappeared after the aptamer-blotting assay, resulting in one
distinct peak for each aptamer (Fig. 2c). Interestingly, in
this step the more prominent peak of A77 at 80°C vanished while the less prominent one at 87°C increased significantly in height. This finding implies that coamplification of two aptamers can result in suppression of
one aptamer. This effect is probably caused by significant
differences in PCR efficiency when amplifying two aptamers of different composition, but is independent of the
aptamer's affinity to the target.
Since A38 showed the highest antiviral activity to VACV in
a preliminary screening, it was chosen for further characterization and binding studies. The A38 sequence is
TACgACTCACTATAgggATCCTgTATATATTTTgCAACTAATTgAATTCCCTTTAgTgAgggTT. Figure 2d
shows the melting profile of the chemically synthesized
A38 as single peak; its two-dimensional structure under
the reaction conditions used as obtained using the m-fold
software [25] is presented in the inset.
In vitro-binding studies of A38
Binding characteristics of A38 to VACV were further validated by dot blot analysis of immobilized VACV particles
stained with biotinylated A38 (OligoService, Berlin, Germany). As shown in Fig. 3a, A38 binds exclusively to
VACV particles and not to the non-infected corresponding
cell culture supernatant or Cytomegalovirus (CMV) particles. Even in high concentrations of 10 nM aptamer, binding is still specific for VACV. The binding to VACV
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particles was concentration dependent and could no
longer be observed for A38 concentrations below 10 pM.
As shown in Fig 3b, Surface Plasmon Resonance Spectroscopy (Biacore) confirmed a significant binding of VACV
particles in suspension to immobilized A38. The binding
was nearly linear for undiluted (red line) and diluted
VACV suspensions (blue line). The slope was proportional to the virus concentration when the drifting baseline caused by aptamer leakage was considered. The linear
slope indicates that the virus concentration was low (109
particles/mL for undiluted virus solution) and far from
the saturation stage. A signal increase could be seen in
spite of the extremely low virus concentration for two reasons: firstly, the SPR signal depends on the refractive index
of the analyte and practically on the mass, which is high
for viruses compared with individual proteins. Secondly,
the virus particle presents the molecular target in multiple
copies on its surface, which leads to enhanced binding to
the immobilized aptamers. Furthermore the results show
that neutravidin-binding does not affect the ability of the
aptamer to bind VACV particles.
Finally, to prove the binding of A38 to VACV with both
binding partners in solution, corresponding to in vivo conditions, Fluorescence Correlation Spectroscopy (FCS) was
performed (Fig. 3c). This technique proved the binding of
A38 to VACV particles by determination of the diffusion
times for tetramethylrhodamine isothiocyanate (TMR)labeled A38 and VACV alone and in combination. In
detail, the respective diffusion times were 36 ± 2 μs for free
TMR (443Da, black line), 165 ± 20 μs for TMR-labeled
A38 (20 kDa, blue line), and 6.5 ± 0.9 ms for TMR-labeled
VACV (350 nm in diameter, green line). When nonlabeled VACV was added to a solution of TMR-labeled
A38, the diffusion time of A38 increased to a magnitude
similar to the one of TMR-labeled VACV (red line), which
was attributable to the binding of A38 to VACV. Control
experiments with CMV (brown line) and 200-nm latex
beads (not shown) mixed with TMR-conjugated A38
showed diffusion times identical to those for TMR-labeled
A38 alone, indicating no binding to both controls. These
data prove that fluorescently labeled A38 binds specifically only to VACV particles, but not to control particles of
similar size.
To prove its biological activity, VACV infection of human
Hep2 cells (Fig. 4), simian Vero E6 cells and primary
chicken embryo fibroblast cells was determined in the
presence of A38 by immunofluorescence assay (IFA). At a
concentration of 2.5 μM, A38 clearly inhibited the spread
of infection in all three cell types. When determining the
cell viability under the influence of A38 at an equal concentration, it caused no increased cell death or apoptosis
as proved by WST-1 cell proliferation assays (WST-1
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column fragment
0
5
10
15
20
25
30
35
40
a)
Signal
Col 2
2400
1800
1200
no VACV
with VACV
relative fluorescence
b)
0
A38
A41
A77
A91
10
20
30
cycle #
600
0
c)
d)
fluorescence (-R'(T))
1800
1200
600
0
1800
1200
600
0
60
65
70
75
80
85
90
temperature /°C
Figure 2
Identification
of high-affinity-binding aptamers by MonoLEX
Identification of high-affinity-binding aptamers by MonoLEX. A combinatorial DNA library (64 b in length) was
applied to an affinity capillary column coated with complete heat-inactivated VACV. Bound aptamers were desorbed from cut
column slices and amplified by real-time PCR. (a) Data derived from different segments along the affinity column show a cumulation of aptamers in distinct segments while other segments do not amplify bound aptamers. Color labels indicate segments
which were used for further evaluation of the aptamer pools. Figure 2b to 2d show the change of fluorescence per change of
temperature plotted versus the temperature. (b) Melting temperature analysis of polyclonal aptamer pools after PCR amplification showing aptamer pools with different nucleic acid composition. In two of the pools (A38 and A77) more than one melting
temperature maximum was observed, indicating different aptamer sub-pools. (c) Melting temperature analysis after aptamer
dot blotting with the target molecule and repeated amplification. In A38 and A77 only one of the two sub-pools was amplified,
indicating that one aptamer is more efficiently amplified after binding. The inset shows the preceding amplification results of an
aptamer blotting assay with VACV (red solid line) and a negative control (black dotted line). The relative fluorescence is plotted vs. the cycle number. (d) Melting profile of the chemically synthesized and further characterized A38 with its predicted
two-dimensional structure.
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a)
CC
VV
CMV
A38
10 nM
1.0 nM
0.1 nM
0.01 nM
resonance units (RU)
VV
VV 1/10
300
16000
200
15600
100
15200
control FC
A38 FC
14800
0
800
resonance units (RU)
16400
b)
0
1600 2400 3200
0
400
800
1200
1600
c)
autocorrelation function
time in s
TMR
A38-TMR
A38-TMR + CMV
VV-TMR
A38-TMR + VV
2.5
2.0
1.5
1.0
10-3
10-2
10-1
100
101
102
correlation time (ms)
Figure
In
vitro-binding
3
studies of A38
In vitro-binding studies of A38. (a) Dot blot of VACV (VR-1536), non-infective cell culture supernatant (CC) and CMV, followed by incubation of biotin-conjugated A38 in various concentrations and labeling with a streptavidin peroxidase conjugate.
(b) Surface Plasmon Resonance measurements (Biacore) of A38. Sequence of injections (injection start): A38 (150 s); VACV
(1050 s); 50 mM sodium carbonate (2320 s); 1:10 diluted VACV (4080 s). The overlaid VACV net binding curves are shown on
the right hand side. (c) Fluorescence Correlation Spectroscopy (FCS). Normalized autocorrelation functions (ACF) of tetramethylrhodamine isothiocyanate (TMR): free in solution (black), coupled to A38 alone (blue), coupled to A38 with added CMV
(brown), coupled to A38 with added VACV (red), and directly coupled to VACV (green). Control experiments with CMV
(brown) and 200-nm latex beads (not shown) mixed with TMR-conjugated A38 showed identical diffusion times to those for
TMR-labeled A38 alone, indicating a binding exclusively to VACV particles.
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Roche Applied Science, Germany) and annexin V/PI flow
cytometric analysis (data not shown).
The antiviral potency of A38 was measured by an endpoint dilution (5 μM, 2.5 μM, 250 nM, 25 nM, 2.5 nM,
250 pM) and immuno-fluorescence-based assay in Hep2
cells against VACV (Fig. 4), followed by determining the
ratio of infected/non-infected cells. A38 inhibited VACV
entry with a 50% inhibitory concentration (IC50) value of
0.59 μM. The antiviral effect exerted by A38 was neither
due to cytotoxicity nor to induced apoptosis. A38 had no
significant effect on cell viability in cytotoxicity assays
(WST-1, Roche, Germany). The compound concentration
at which uninfected Hep2 cell proliferation was inhibited
by 50% (CC50) was > 10 μM, which was above the available synthesized concentration for A38. This resulted in a
selective index (SI) of > 16.95 (SI = CC50/IC50). A similar
antiviral activity of A38 could be detected against a broad
spectrum of OPV-like Cowpox virus, Ectromelia virus and
Camelpox virus as shown by IFA, by quantitative real-time
PCR of viral DNA used as described previously [26] and
by virus titration of the corresponding supernatants (Fig.
5). Reduced virus proliferation could be confirmed in all
experiments. Taken together, these results demonstrate
that A38 binds to a target common to all OPV, is a potent
and specific inhibitor of OPV. However, the inhibition of
Ectromelia virus proliferation was less pronounced.
c)
Discussion
When the first aptamers were developed 15 years ago, it
was predicted that they had an enormous potential as a
feasible alternative to antibodies. Although the application of antibodies for the detection and therapy of infectious agents is well established, aptamers have some
potential advantages over antibodies that may fill the gaps
that antibody-based applications possess and that expedite the use of aptamers in the virologist's laboratory [7].
These advantages include their small size, eased cell penetration, rapid and cheap synthesis including a variety of
chemical modifications and the fact that aptamers are
non-immunogenic. Probably their crucial benefit is a
selection process performed in vitro. Thus, aptamers can
50
b)
d)
proportion of positive control
a)
Since a potential therapeutic use of A38 in OPV infections
requires sufficient nuclease resistance of the DNA
aptamer, a precondition that unmodified RNA aptamers
often fail to meet, its stability was evaluated by real-time
PCR as described above. For that purpose, A38 was incubated in serum for 24 h at 37°C. As shown by the nearly
identical CT values for A38 in water and in serum, no significant degradation of A38 was detected by quantitative
PCR after a 24-h incubation in serum (Fig. 6). A38 still
inhibited virus dissemination after 7 days in medium to a
degree similar to fresh A38, as confirmed by IFA (data not
shown).
40
IFA
PCR
Titer
30
20
10
0
DW
1/02 XV Call
V LE
VAC VACV I-H CPXV 8
CP
Figure
In
vitro antiviral
4
activity of A38
In vitro antiviral activity of A38. Immunofluorescence
microscopy of VACV-infected Hep2 cells incubated with A38
after 24 hours. (a) Cells infected with VACV at MOI of 0.1,
(b) cells incubated with a mixture of VACV and 2.5 μM A38
or (c) VACV and 2.5 μM of the complete template library.
(d) Control of non-infected cells. Bar, 100 μm. Red cells are
counterstained with Evans's Blue.
P
CAM
ECT
Figure 5activity of A38 against various OPV
Antiviral
Antiviral activity of A38 against various OPV. OPV
were propagated in cell culture in presence or absence of
A38 as described above. The degree of infection in comparison to a positive control in the absence of A38 was determined by enumerating the number of infected cells (IFA,
black bars), quantification of intracellular poxvirus DNA by
real-time PCR (red bars) or determination of the poxvirus
titer in the corresponding supernatant (green bars).
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Figure 6of DNA aptamer A38
Stability
Stability of DNA aptamer A38. A38 was incubated for 24 h at 37°C in water or, in comparison, in serum. No significant
shift of CT value was observed by quantitative real-time PCR for the serum-incubated aptamer (brown lines, n = 3) in comparison to the fresh aptamer A38 (blue lines, n = 2).
be selected against targets that are either weakly immunogenic or toxic, like toxin proteins [27]. These targets are
usually not applicable for in vivo production of monoclonal antibodies in mice; however, highly affine aptamers have been selected against some of these problematic
targets [5]. Moreover, the chemical and physical conditions for aptamer selection can be adapted to the real environment in which the aptamer will finally be applied. This
includes cross-selection against similar targets that have to
be excluded from an aptamer's detection pattern.
With the FDA approval of the first aptamer-based drug
Macugen and the recent publication of an aptamer-based
cocaine sensor, aptamers are starting to tap their full
potential with the increasing need for additional specific
detection tools.
Most of the aptamers developed have been selected by
SELEX or modifications of SELEX. In this study we evaluated a new selection procedure for aptamers. As demonstrated for the selection of OPV-specific DNA aptamers,
the MonoLEX method is a straightforward procedure for
the identification of high-affinity aptamers to a target
structure of interest. Although SELEX has proven to generate specific aptamers for several targets, MonoLEX can be
a valuable alternative for aptamer selection. Based on one
affinity chromatography, an optimized enrichment of
high-affinity aptamers is obtained by physical segmentation of the affinity resin.
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The affinity of the bound aptamers is expected to decrease
slightly in the direction of the outlet of the column. However, practical experience shows that the oligonucleotides
assort in distinct clusters along the column. Column segmentation allows a direct desorption of high-affinity
aptamers from individual column fragments, preventing
their possible loss due to an amplification efficiency lower
than that of low-affinity binders. The combination of differently coated affinity chromatography columns allows
the depletion of aptamers that may also bind to structures
closely related to the target, therefore increasing the specificity of the aptamers selected.
As shown by several analytical techniques, the selected
DNA aptamer A38 possesses a considerable stability in
binding to VACV. This binding could not only be demonstrated with immobilized virus particles on a dot blot, but
also vice versa with immobilized aptamers as shown by
Surface Plasmon Resonance Spectroscopy, a situation
found on detection sensors for poxvirus particles. As
described among others for HCV [28], A38 could be
immobilized on sensor surfaces to identify poxvirus particles in complex matrices, as frequently seen in samples
suspected to be of bioterrorist origin. The development of
such a sensor has already been considered. Concerning
the therapeutic application of A38, it was also important
to prove its binding to OPV with both partners in solution. To this end, we performed FCS studies and demonstrated the binding of A38 to VACV particles, but not to
CMV or latex beads of similar size. While FCS proved to
be a reliable and sensitive method to demonstrate
aptamer-virus interactions in solution, the binding of A38
to VACV-infected cells could not be observed by confocal
fluorescence microscopy. The reason for this failure is still
not clear but may be a problem of sensitivity. Although
the binding partner of A38 on OPV particles is still
unknown, it must be highly conserved and present on all
different OPV. The in vitro replication of several OPV
could significantly be inhibited in the presence of A38 in
a concentration-dependent manner, with IC50 values of
0.59 μM, a concentration that is in agreement with previously published aptamers specific for other viruses [17].
Further studies have to evaluate the mechanism of inhibition and the potential to inhibit OPV replication in animal models.
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aptamers. Especially in scenarios where infections with
new, emerging pathogens have to be detected or treated, a
fast technique providing specific detection tools may be
helpful. The spectrum for different applications of aptamers like A38, either on a sensor for OPV detection or for
treatment of infections, requires an extremely high stability of the aptamer. The use of DNA aptamers facilitates
such a high stability without modifications, even in body
fluids.
With the improving automation of the aptamer selection
process and the growing knowledge of pathogen-specific
targets, the number of aptamers used in diagnostics and
therapy of infectious diseases will dramatically increase in
the future.
Methods
Virus preparation
Vaccinia virus (VACV, strain NYCDH, ATCC #VR-1536),
VACV (strain Lister Elstree), cowpoxvirus (CPXV, strain
81/02), mousepoxvirus (ECTV strain Nü-1) and camelpoxvirus (CMLV, strain CP19) were propagated in Hep2
cells (ATCC CCL-23) at a multiplicity of infection (MOI)
of 0.25 following standard procedures with Dulbecco's
Modified Eagle Medium (DMEM) containing 1 g/L D-glucose, L-glutamine and 25 mM HEPES buffer. Infected cells
were incubated for approximately 4 days until a pronounced cytopathic effect was observed. Virus-containing
supernatant and cells were harvested and subsequently
separated by centrifugation (10 min at 1000 × g) after
freeze thawing. Virus-containing supernatant was titered
according to Reed and Münch [29] and stored at -75°C
until use. For inactivation prior to selection, the virus suspension was heat-treated at 60°C for 1 h and checked for
infectious activity by cell culture as described above. As
control virus for binding experiments, CMV was kindly
provided by Stefanie Thulke, Charité Berlin, Germany.
Conclusion
Selection of high-affinity-binding aptamers by MonoLEX
A MonoLEX selection process comprised the following
steps: Chemical coupling of the target (VACV) to the affinity column, incubation with the combinatory library,
extensive washing to elute non-binding oligonucleotides,
physical segmentation of the affinity column, PCR amplification of the aptamers bound to different column fragments and finally an aptamer dot blot assay to evaluate
the binding specificity.
The need for specific diagnostic or therapeutic tools in
infectious diseases is obvious. Here we present a new
selection approach for aptamers, called MonoLEX, and
describe the selection of an OPV-specific aptamer. According to MonoLEX, the amplification of aptamers from
affinity-column segments after complete depletion of
low-affinity binders by affinity chromatography might be
a fast and reliable technology for isolation of highly affine
The selection experiments started from a combinatory
library that was 64 bases long (5'-TACGACTCACTATAGGGATCC-N7-A-N7-A-N6-GAATTCCCTTTAGTGAGGGTT3') including 20 random nucleotide positions and two terminal PCR primer sequences diluted in DMEM. For selection, an affinity capillary column was coated with heatinactivated VACV (strain NYCDH) particles. To reduce
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unspecific binding, the complete template library was first
applied to an affinity capillary column covalently coated
with the cell culture supernatant of non-infected Hep2
cells. After application of the remaining combinatory
library to the selection column, low-binding oligonucleotides were washed off with approximately the 5000-fold
column volume of Tris buffer (pH 7.3, 0.1% TWEEN-20),
DMEM (0.1% TWEEN-20) and phosphate buffer (pH 7.5,
0.1% TWEEN-20). The column was then cut into slices.
Target-bound aptamers were desorbed from the column
slices by denaturation of the target and amplified by
quantitative real-time PCR on an iCycler (BioRad,
Munich, Germany), using 2 pmol of the primers AP7
TACgACTCACTATAgggATCC and AP3: AACCCTCACTAAAgggAATT (Metabion, Martinsried, Germany) in
a SybrGreen Mastermix (Cambrex, NJ, USA). The mixture
of such amplified aptamers is called "polyclonal aptamers". Cycling conditions were as follows: Initial denaturation at 95°C for 5 min and 40 repeats of denaturation at
95°C for 15s, primer annealing at 50°C for 30s and elongation at 68°C for 30s. Subsequently to PCR, a fluorescence curve melting analysis (FCMA) was performed,
cooling down the PCR reaction product to 50°C and
increasing the temperature with a maximum ramping rate
up to 90°C. Fluorescence was monitored continuously
and melting peaks were calculating with the iCycler software.
Pyrosequencing
Pyrosequencing was performed with a PyroMark ID System (Biotage, Sweden) using the Pyro Gold kit. Briefly,
aptamer candidates were amplified by PCR as described
above, applying one biotinylated primer AP3. Singlestranded DNA was prepared by denaturation in NaOH
and binding to sepharose beads as recommended by
Biotage. The single-stranded DNA was sequenced with
primer AP7 in the sequencing mode of the PyroMark ID
System. Usually, the complete aptamer sequence could be
determined unambiguously.
Aptamer synthesis
Aptamer A38 was purchased as lyophilized oligonucleotide (Oligoservice, Berlin, Germany), either non-modified, 5' biotinylated or 5' tetramethylrhodamine
isothiocyanate (TMR) labeled. All aptamers were reconstituted in Tris-HCL (10 mM, pH 8.4) as 100 μM stock solutions and stored at -20°C until use for further
experiments.
Aptamer blotting assay
To validate the presence of target specific aptamers in the
aptamers derived from the individual slices of the affinity
column, a PCR-enhanced dot blot assay was performed.
Briefly, the target VACV particles were chemically immobilized to a polypropylen tube. Polyclonal aptamers were
http://www.biomedcentral.com/1472-6750/7/48
purified by agarose gel electrophoresis from PCR mastermix components and incubated in the tubes overnight.
After ample washing the immobilized aptamers were
amplified by quantitative PCR as described above.
Dot blot
A38 was tested at various concentrations against VACV,
non-infective cell culture supernatant and CMV. 30 μL of
sucrose-cushion-purified VACV (~107 particles) were
spotted onto a 0.2 μm PVDF membrane (Schleicher &
Schuell, Dassel, Germany). After incubation with blocking buffer (PBS, 0.1% Tween 20 und 10% low-fat milk
powder), the membrane was incubated with varying
amounts of biotin-conjugated A38 (0.01–10 nM) for 1 h
at RT. Following repeated washing with PBS and incubation with a streptavidin peroxidase conjugate (Dianova,
Hamburg, Germany) for 1 h, the detection was performed
by chemiluminescence using SuperSignal® West Pico
(Pierce, USA) according to the manufacturer's instructions.
Fluorescence Correlation Spectroscopy (FCS)
The binding of the aptamer to VACV was assayed by Fluorescence Correlation Spectroscopy (FCS)[30]. We used a
confocal microscope ConfoCor (Carl Zeiss, Jena; Evotec
Biosystems, Hamburg, Germany) and analyzed the diffusion of dye-labeled monomers and complexes by the normalized autocorrelation function (ACF), as described
previously [31]. The fluorescence fluctuations of the
marker dye, tetramethylrhodamine isothiocyanate
(TMR), were recorded for 5 min at RT and submitted to
autocorrelation analysis using the hardware and software
by Evotec Biosystems (Hamburg, Germany). The autocorrelation function was analyzed in terms of particle diffusion through the confocal volume and the intrinsic triplet
state dynamics of the fluorophor TMR [32]. The analysis
yielded the number of particles and the mean dwell time
(diffusion time) of each particle species in the confocal
volume. Total particle numbers between 0.5 and 1.5 were
observed. Data were rescaled to a total particle number of
one for better comparison. The calculated diffusion times
were characteristic for TMR, A38-TMR and VACV-TMR.
Saturation of the binding of TMR-labeled A38 to nonlabeled VACV was indicated by a complete shift of the
autocorrelation function (ACF) from a shorter to a longer
correlation time characteristic for VACV-TMR. The following concentrations were applied: 3 nM TMR, 5 nM A38TMR, 108 PFU/ml VACV-TMR, 108 PFU/ml non-labeled
VACV and 108 PFU/ml non-labeled CMV in PBS buffer.
Surface Plasmon Resonance measurements (Biacore)
Binding experiments were done with the SPR-based
instrument Biacore™2000 (Biacore, Freiburg, Germany)
and sensor chips CM4, using the control software version
2.1 and evaluation software version 3.0 (Biacore AB, Upp-
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BMC Biotechnology 2007, 7:48
sala, Sweden). The running buffer was PBS containing
0.005 % (v/v) Tween 20. This buffer was also used for
sample dilution. The temperature of the flowcells was
25°C.
For the immobilization of Neutravidin, the carboxymethyl dextrane matrix of the chip was activated for 7 min
with 35 μl of a mixture of 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and
0.05 M N-hydroxysuccinimide (NHS). Neutravidin
(Pierce, Rockford, USA) was diluted to 20 μg/ml in 10
mM phosphate pH 5.5 and injected into the activated
flowcell 2 for 5 minutes. Non-reacted sites were blocked
with 1 M ethanolamine pH 8 for 1 min. The amount of
immobilized Neutravidin was 2100 resonance units (RU)
for flowcell 2. The control flowcell 1 was only activated
and blocked.
Binding experiments were performed with aptamer A38
that was injected into flowcell 2 at a concentration of 1
μM in PBS for 5 min, yielding 480 RU for bound aptamer.
After 5 min of washing with buffer, the dissociation of
aptamer was constantly slow, and the virus suspension
(109 particles/ml) was injected into flowcells 1 and 2 for 5
min. Bound particles were then eluted with 50 mM
sodium carbonate for 1 min. Finally, a tenfold diluted
virus suspension was injected (Fig. 3b, left panel). Specific
binding sensorgrams were obtained by normalization to
the curve of the control flowcell 1. The background was set
to zero, and the injection start was synchronized (Fig 3b,
right panel). Kinetic evaluation of the binding was not
possible, as the binding of virus particles is a multisite
attachment to the aptamer layer.
Immunofluorescence Assay (IFA)
5 × 103 Hep2 cells were propagated in 96-well plates
(Nunc, Wiesbaden, Germany) for 24 h at 37°C. Cells were
either infected with VACV at a MOI of 0.1 alone or
together with 2.5 μM A38 or 2.5 μM of the combinatory
library. Cells were incubated for another 24 h at 37°C. For
visualization OPV-infected cells were fixed in 4% formalin and stained with a polyclonal human anti-pox IgG
(1:500, Omrigam, USA), followed by a FITC-conjugated
goat anti-human IgG (1:50, Caltag Laboratories, USA)
according to standard procedures and contrasted by Evans
Blue staining. The number of infected cells was determined by fluorescence microscopy. Briefly, four representative pictures ((??)) were taken. The total cell number,
stained with Evans Blue, and the number of infected cells,
stained with the anti-poxvirus serum, were enumerated by
application of the software package ImageJ http://
rsb.info.nih.gov/ij/.
http://www.biomedcentral.com/1472-6750/7/48
Authors' contributions
AN designed and coordinated the study and drafted the
manuscript. AKu performed cell culture, participated in
FCS, Dot Blot and IFA work and drafted the manuscript.
AD and DM performed cell culture, Dot Blot and IFA
work. OP and WJ initiated work with FCS and helped to
draft the manuscript. HS performed FCS work. FS and WS
initiated and performed work with Biacore. AKa helped to
design the study, performed aptamers blotting assay and
helped to draft the manuscript. CD performed the aptamers selection. GP participated in the study design and
coordination.
Acknowledgements
Parts of the study were supported by the German Federal Ministry of
Health (Foko 1-121-24461), the city of Berlin and EFRE (NanoMed, No.
EFRE 20002006 2ü/2) and German Federal Ministry of Education and
Research (BioHyTec, No 03i 1308). The authors are grateful to Daniel
Stern for preparation of figure 1 and Ursula Erikli for proof-reading the
manuscript.
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